Composition comprising highly-concentrated α1 proteinase inhibitor and method for obtaining thereof

ABSTRACT

Compositions include highly-concentrated Alpha-1 Proteinase Inhibitor (A1PI) in a concentration greater than or equal to 100 mg/ml. Pharmaceutical compositions can be prepared from these compositions. The pharmaceutical compositions can be suitable for subcutaneous administration. The highly-concentrated A1PI solutions can be obtained by single-pass tangential flow filtration (SPTFF).

BACKGROUND Field

The present disclosure is related to the field of pharmaceuticalproducts. Certain embodiments herein relate to compositions comprisinghighly-concentrated Alpha-1 Proteinase Inhibitor (A1PI), which can beused for many therapeutic indications, and methods for obtaining thecompositions.

Description of the Related Art

Alpha-1-Proteinase inhibitor (A1PI), also known as Alpha-1-Antitrypsin(AAT) or α₁-Antitrypsin, is a proteinase inhibitor that acts on avariety of cellular proteases A1PI plays a major role in tissuehomeostasis through the inhibition of neutrophil elastase action, andthrough other mechanisms.

Congenital deficiencies of A1PI allow uncontrolled activity ofneutrophil elastase and the subsequent degradation of dastin, anessential protein that confers elasticity to tissues, particularly thelungs. The absence of elastin may result in respiratory complicationssuch as pulmonary emphysema and hepatic cirrhosis.

Chronic intravenous (IV) administration of A1PI to treat AAT deficiencyis burdensome, requires professional assistance (administered in thepatient's home or in clinics, hospitals, etc), and can cause immediatehypersensitive reactions. To overcome said problems, a new concentrationprocess and a new formulation have been developed by the inventors for anovel product which comprises highly concentrated A1PI. The concentratedformulation detailed in the invention enables a broader spectrum ofparenteral administration, which may include intravenous, subcutaneous,aerosol, and intradermal administration. This product could satisfy thelong-standing unmet need to make dosing easier to administer by patientsat home, without a supporting healthcare professional, thus reducingtreatment costs, and is suitable for chronic treatment.

The inventors of the present invention are not aware of any prior artmethod for obtaining A1PI with a concentration higher than 100 mg/ml.Attempts to increase the concentration of A1PI using conventional flatcassette tangential flow filtration (TFF) results in generation ofunacceptable high level of aggregates even using small amount of buffersalts. The present inventors have surprisingly found that using amodified tangential flow ultrafiltration method that used the samemembrane type and molecular weight cut-off (MWCO) as the conventionalultrafiltration/diafiltration (UFDF) step, but operated at reduced flowrates, higher transmembrane pressure (TMP), and larger membrane area toincrease flow-path length, could concentrate A1PI to at least 10% (w/v,100 mg/ml) in a single-pass tangential flow filtration (STIFF) after ithad been diafiltered against water to remove all process salts. Thepresent inventors have surprisingly found that, using a further step ofSPITF against water for injection (WTI), it is possible to obtain aconcentration of A1PI of at least 100 mg/ml, and said solution can besubsequent formulated with uncharged excipients to achieve isotonicconditions. This enabled formulation of a concentrated A1PI withuncharged excipients to adjust osmolality, while addressing the previouspoor stability performance with charged excipients.

SUMMARY

An embodiment of the present disclosure provides a compositioncomprising Alpha-1 Proteinase Inhibitor (A1PI) in an aqueous solution,wherein the concentration of A1PI is greater than or equal to 100 mg/ml,preferably is greater than or equal to 150 mg/ml, more preferably isgreater than or equal to 200 mg/ml.

In some preferred embodiments, the composition comprising A1PI furthercomprises one or more uncharged excipients. The term “unchargedexcipients” mean that no net charge of one or greater is present at nearneutral pH with said excipients.

In some preferred embodiments, the one or more uncharged excipients areat a concentration necessary to achieve isotonicity, i.e., between 220and 410 mOsm/kg H₂O, preferably about 300 mOsm/kg H₂O.

In some preferred embodiments, the one or more uncharged excipients areselected from the list consisting of amino acids, sugars, and polyols,including sorbitol, serine, trehalose, alanine, sucrose, and mannitoland combinations thereof. More preferable, the one or more unchargedexcipients are alanine, sorbitol or trehalose and combinations thereof.

In some preferred embodiments, said uncharged excipient(s) are in thecomposition at a concentration of approximately 120 mM, to achieveacceptable osmolality.

In some embodiments, the pharmaceutical composition can be administeredto a patient in need thereof.

Another embodiment of the present disclosure provides a pharmaceuticalcomposition comprising the above-mentioned composition and apharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is formulated forintravenous, subcutaneous, aerosol, or intradermal administration,preferably for subcutaneous administration.

In some embodiments, the pharmaceutical composition is encapsulated innanoparticles or it comprises a time-release polymer.

Another embodiment of the present disclosure provides a method forpreparing the above-mentioned composition, comprising preparing asolution of A1PI by concentrating an initial solution of A1PI by SPTFF.

In some embodiments, a method for preparing a solution of A1PI by SPTFFwherein the final concentration of A1PI is at least 100 mg/ml,preferably at least 150 mg/ml, more preferably at least 200 mg/ml.

In some embodiments, a method for preparing a solution of A1PI by SPTFFis carried out against water for injection (WFI).

In some embodiments, a method for preparing a concentrated solution ofA1PI comprises after the SPTFF step a formulation with one or moreuncharged excipients selected from the list consisting of sorbitol,serine, trehalose, alanine, sucrose, and mannitol, and combinationsthereof. More preferable, the one or more uncharged excipients arealanine, sorbitol or trehalose and combinations thereof.

pH is controlled to near neutral (around 6.6 to 7.4) without the use ofbuffers, but through adjustment upon addition of formulationexcipient(s), and remains stable throughout storage.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an embodiment of a method in which a SPTFF step and aformulation are added to a prior art process.

FIG. 2 shows B₂₂ vs. excipient type from self-interacting chromatography(SIC) experiments.

FIG. 3 shows the aggregate percentage vs. days for A1PI 20% formulationsat 40° C.

FIG. 4 shows the aggregate percentage measured by size exclusionhigh-performance liquid chromatography (SE-HPLC) in the liquidformulations of A1PI stored in vials at 5° C.

FIG. 5 shows the Zeta Potential vs. pH of different formulations of A1PIat 0.12 M.

DETAILED DESCRIPTION

Currently, A1PI solutions are commercially available (Prolastin-C,Grifols; Glassia, Shire; Zeinaira, CSL; Aralast, Baxter) to treat humancongenital deficiency of the protein (Alpha-1 Antitrypsin Deficiency,AATD). One limitation that all these products have in common is thatthey contain A1PI at relatively low concentrations (about 20 to 50mg/ml). For this reason, their only suitable route of administration asa therapeutic has been weekly intravenous injection.

Chronic intravenous administration of A1PI to treat AATD is burdensome,requires professional assistance, and can cause immediate hypersensitivereactions. Therefore, there is a longstanding unmet need to make dosingeasier to administer by patients at home without an assisting healthcareprofessional, which would considerably reduce treatment costs and wouldmake it suitable for chronic treatment. In order to enable a wider rangeof parenteral administration routes, the concentration of A1PI in theproducts should be increased. However, it is not possible to producestable concentrated liquid A1PI based on the current methods of A1PIpurification, with existing formulations.

Surprisingly, the inventors found that including an additional step ofSPTFF at the end of purification allowed the production ofhighly-concentrated A1PI of at least 100 mg/ml, preferably at least 150mg/ml, and most preferably 200 mg/ml. The novelty of this step of theprocess is that it had to be performed in the absence of buffers andsalts, in the presence of WFI and formulated later by addition of one ormore uncharged excipients selected from the list consisting of sorbitol,serine, trehalose, alanine, sucrose, and mannitol, and combinationsthereof, more preferably alanine, sorbitol or trehalose and combinationsthereof. Importantly, the resulting compositions are suitable for humanadministration since they comply with the values of osmolality,stability, and viscosity required by the regulatory agencies.

Compositions A1PI

In some embodiments, the present disclosure provides a compositioncomprising A1PI. In some embodiments, the composition comprises A1PI inan aqueous solution, in some embodiments, the concentration of A1PI inthe composition is at least 100 mg/ml. In some embodiments, theconcentration of A1PI in the composition is 150 mg/ml, in someembodiments, the concentration of A1PI in the composition is ≥200 mg/ml.In some embodiments, the concentration of A1PI in the composition isabout 100, 140, 180, 220, 260, 300, 340, 380, 420, 460, or 500 mg/ml, orwithin a range defined by any two of the aforementioned values.

Pharmaceutical Compositions of A1PI

In some embodiments, a pharmaceutical composition is provided. In someembodiments the pharmaceutical composition comprises an A1PI solution.In some embodiments, the concentration of A1PI in the solution is atleast 100 mg/ml.

In some embodiments, the concentration of A1PI in the solution is about100 mg/ml to about 500 mg/ml. In some embodiments, the concentration ofA1PI in the solution is about 100, 150, 200, 300, 400, or 500 mg/ml, orwithin a range defined by any two of the aforementioned values.

In some embodiments, the osmolality of the solution is about 220 mOsm/kgto about 410 mOsm/kg. In some embodiments, the osmolality of thesolution is about 220, 240, 270, 300, 330, 360, 390 or 410 mOsm/kg, orwithin a range defined by any two of the aforementioned values.

In some preferred embodiments, the pharmaceutical composition furthercomprises one or more uncharged excipients selected from the groupconsisting of amino acids, sugars, and polyols, including sorbitol,serine, trehalose, alanine, sucrose, and mannitol, and combinationsthereof, more preferably alanine, sorbitol or trehalose and combinationsthereof.

pH is controlled to near neutral (around 6.6 to 7,4) without the use ofbuffers, but through adjustment upon addition of formulationexcipient(s), and remains stable throughout storage.

In some preferred embodiments, the pharmaceutical composition comprisesone or more uncharged excipients at a final concentration of about 120mM to achieve acceptable osmolality.

One or more embodiments of the pharmaceutical composition providedherein can be administered to a patient in need thereof in someembodiments, the pharmaceutical composition is administeredintravenously, intradermally, subcutaneously, intramuscular, orally, ora combination thereof.

In some embodiments, other non-limiting routes of administrations arecontemplated, for example, parenteral, intraarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracelebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,intralesional, bolus, vaginal, rectal, buccal, sublingual, intranasal,or transdermal.

In some embodiments, the pharmaceutical compositions provided hereincomprise active ingredients, inactive ingredients, excipients, and/orpharmaceutically acceptable carriers. A wide variety of pharmaceuticallyacceptable carriers are available and well-known in the art. Theformulation of pharmaceutical compositions is determined in part by theparticular composition being administered as well as by the particularmethod and/or route used to administer the composition.

Pharmaceutical compositions can comprise aqueous and non-aqueous,isotonic sterile injection solutions, which can comprise antioxidants,buffers, bacteriostats, and solutes that render the composition isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives.

In some embodiments, one or more embodiments of the pharmaceuticalcomposition provided herein are used to treat one or more of respiratorycomplications such as pulmonary emphysema, chronic obstructive pulmonarydisease, etc. In some embodiments, one or more embodiments of thepharmaceutical composition provided herein are used to treat hepaticcomplications such as hepatic cirrhosis. In some embodiments, one ormore embodiments of the pharmaceutical composition provided herein areused to treat any disease or condition related to A1PI deficiency, orbenefited by augmentation with A1PI.

Methods of Preparing Concentrated A1PI Solution

In some embodiments, a method of preparing a solution of A1PI isprovided. In some embodiments, the method comprises concentrating asolution of A1PI in water or with uncharged excipients by conventionalflat cassette tangential flow ultrafiltration (TFF). In someembodiments, the final concentration of A1PI in the concentratedsolution is as much as 100 mg/ml.

In some embodiments, the method comprises concentrating a solution ofA1PI in water or with uncharged excipients by SPTFF, where the finalconcentration of A1PI in the concentrated solution is about 150 mg/ml toabout 500 mg/ml. In some embodiments, the final concentration of A1PI inthe concentrated solution is about 150, 180, 200, 220, 260, 300, 340,380, 420, 460, or 500 mg/ml, or within a range defined by any two of theaforementioned values.

In some preferred embodiments, the solution of A1PI in WFI is obtainedby SPTFF.

In some embodiments, a method for preparing a concentrated solution ofA1PI comprises, after the SPTFF step, a formulation with one or moreuncharged excipients selected from the group consisting of amino acids,sugars, and polyols, including sorbitol, serine, trehalose, alanine,sucrose, and mannitol, and combinations thereof, more preferablyalanine, sorbitol or trehalose and combinations thereof.

pH is controlled to near neutral (around 6.6 to 7.4) without the use ofbuffers, but through adjustment upon addition of formulationexcipient(s), and remains stable throughout storage.

In some preferred embodiments, the concentration uncharged excipient inthe composition is about 0.12 M, or sufficient to adjust osmolality toisotonicity.

Additional Embodiments

Thus, in some embodiments, alternate methods of administration areemployed for such a concentrated A1PI formulation. For example,nano-encapsulation (i.e., encapsulation in nanoparticles) withtime-release polymer for intradermal dosing. Such time release polymersfor nano-encapsulation are well-known to those skilled in the art. Forexample, Tran Thi Dat Nguyen (2015), “Synthesis of timed-release polymernanoparticles,” of the Australian Institute for Bioengineering andNanotechnology at the University of Queensland, available in the espacelibrary of the University of Queensland under DOI 10.14264/4.2015.605describes the use of nanoparticles self-assembled from randomthermoresponsive copolymers, such as copolymers of PNIPAM and PDMAEA.The complete disclosure of this article is hereby incorporated by thisreference thereto. Other methods are also applicable to achieve safedosing of highly concentrated A1PI.

EXAMPLES Comparative Example 1

High Concentration A1PI Process Flow According to the Prior Art.

Alpha-1 MP (U.S. Pat. No. 6,462,180 B1), Liquid Alpha (U.S. Pat. No.9,616,126 B1), and Alpha-1 HC (US 20110237781 A1) processes make A1PI upto 50 mg/ml using a typical recirculating (TFF) UF step to concentrate,followed by a diafiltration (DF) step with water to remove buffer saltsto prepare bulk for final formulation and adjustment to 50 mg/ml ofprotein. The formulation consists of a 2.0 mM sodium phosphate buffer tomaintain pH, and either a salt (Alpha-1 MP and Alpha-1 HC; 100 mM or 150mM NaCl, respectively) or amino acid (Liquid Alpha; 200 to 300 mMalanine) to adjust osmolality to isotonic conditions of 220-410 mOsm/kg.Likewise, other A1PI formulations are similarly prepared (Table 1).

TABLE 1 A side by side comparison of several A1PI preparations, withconcentration and formulations. Alpha Grifols; Kamada; CSL; Therapeutics2002 ARC; 2011 2012 1999 U.S. Pat. No. 2000 U.S. Pat. No. U.S. Pat. No.U.S. Pat. No. 6,462,180 U.S. Pat. No. 7,879,800 8,124,736 5,981,715Prolastin-C 6,093,804 Glassia Zemaira Aralast 50 mg/ml; 10-20 mg/ml;20-40 mg/ml; 50 mg/ml; 20 mg/ml; 0.02M NaP, 0.02M NaP, 0.02M NaP, 0.02MNaP, 0.02M NaP, 0.1M NaCl, 0.1M NaCl 0.1M NaCl, 0.045M NaCl 0.1M NaCl,pH 6.6-7.4 pH 6.8-7.0 pH 6.5-7.5 3% mannitol, pH 8.0 pH 6.6-7.4

As explained above, the process of the present invention incorporatestwo additional steps to the above process: SPTFF concentration andformulation, as shown in FIG. 1 .

Example 2

Evaluation of B₂₂ Value as a Indicator of Aggregation in the UnchargedExcipients A1PI Solutions Obtained with the Process of the PresentInvention.

The process of the present invention involves a SPTFF concentration withWFI and a formulation with uncharged excipients. Several of theuncharged excipients at 0.12 M concentrations, pH 7.0, as well as low pHor high salt formulation controls, were used to make 20% A1PI solutionsand applied to Self-interacting chromatography (SIC) columns produced byconjugating A1PI to a Toyopearl AF-formyl-650M (Tosoh Biosciences)resin, and the retention time recorded. The retention time of theprotein was converted to the osmotic second virial coefficient (B₂₂),which is a measure of protein-protein interactions. FIG. 2 presents aplot of B₂₂ vs excipient type. The higher the B₂₂ value the greater theprotein-protein repulsion (preferred to minimize aggregation) (Payne etal., “Second Virial Coefficient Determination of a Therapeutic Peptideby Self-Interaction Chromatography” Biopolymers (Peptide Science), Vol.84, 527-533 (2006)). The lowest B₂₂ values were predictably observed forthe low pH negative control (0.12 M KCl, pH 6.0) and the formulationcontrol (20 mM sodium phosphate, 75 mM NaCl, pH 7.0), conditions whichwere known to be the least favorable (highest protein-proteininteraction) for A1PI. Sorbitol, serine trehalose, alanine and mannitolall had higher B₂₂ values, indicating more protein repulsion, withmannitol having the highest B₂₂ value. WFI solutions, which relied onintrinsic charge repulsion at pH 7.0, had intermediate B₂₂ values. Theuncharged excipients were at 0.12 M and pH 7.0.

Example 3

Evaluation of A2PI Aggregation in the Uncharged Excipients.

Thermal kinetics studies of 20% A1PI formulations at 40° C. measuredaccelerated aggregation by SE-HPLC over time. A1PI 20% solutions ofvarious 0.12 M excipient formulations (mannitol, alanine, serine,sorbitol, and trehalose) at pH 7.0, along with the control formulation(16 mM sodium phosphate, 60 mM NaCl, pH 7.0), were incubated at 40° C.for 7 days and analyzed by SE-HPLC (FIG. 3 ). These data grouped inthree categories, with the control formulation having the highestaggregation rate, as expected, while mannitol, alanine, serine andsorbitol had an intermediate aggregation rate, and trehalose had asignificantly lower aggregation rate than the others. In conclusion, theresults show that less aggregation occurred in the presence of unchargedexcipients, compared with the control.

Example 4

Evaluation of A1PI Stabilily in the Presence of Uncharged Excipients.

On the other hand, the aggregation was measured by SE-HPLC over time andat different A1PI concentrations in the presence of charged anduncharged excipients. A1PI solutions of FIG. 4 show the aggregationpercentage in vials stored at 5° C. A1P1 at 50 mg/mL (diamonds) and A1PIat 200 mg/mL (circles) with salt to control osmolality, and A1PI at ˜200mg/mL formulated in only 0.12M trehalose (triangles) show differentrates of aggregation. Similar to what Bauer (U.S. Pat. No. 7,879,800)showed in Table 12, the 20% A1PI formulated in 20 mM sodium phosphate 75mM sodium chloride, pH 7.0 had a very high rate of aggregation, comparedto A1PI in similar excipients at 5%. However, the 20% A1PI formulatedwith only 120 mM trehalose, pH 7.0 showed much less aggregation underidentical storage conditions.

Example 5

Daily or Weekly Dosing of A1PI for a 70 kg/100 kg Patient with EitherSubcutaneous (SC) or Intravenous (IV) Administration.

It is understood that subcutaneous dosing is volume constrained, withsingle-site injections often limited to about 25 mL. Therefore, a higherconcentration of A1PI is needed to achieve dosing to prescribed amounts.Current dosing to 60 mg/kg can be achieved, with a 1.2 absorptioncoefficient (EP 2,214,699 B1), with weekly dosing of 15% A1PI at 2 sitesor with 20% A1PI at a single site for the average patient (Table 2).

TABLE 2 Daily or weekly dosing of Alpha-1 HC for a 70 kg/100 kg patientwith either subcutaneous (SC) or intravenous (IV) administration wouldbe most feasible with concentrations at or above 150 mg/ml. Daily SCWeekly SC Dosing Weekly IV Dosing (ml)* (ml)* Dosing (ml)* A1PI 70 kgpatient / 70 kg patient / 70 kg patient / Concentration 100 kg 100 kg100 kg (mg/ml) patient patient patient 50 14.4 / 20.6 100.8 / 144 84 /120 100 7.2 / 10.3 50.4 / 72 42 / 60 150 4.8 / 6.9 33.6 / 48 28 / 40 2003.6 / 5.1 25.2 / 36 21 / 30 *Assuming a 120% multiplier for SCabsorption adjustment.

Example 6

Highly Concentrated A1PI by SPTFF.

A method to achieve compositions comprising highly concentrated A1PI,which could be used for many therapeutic indications, includes theapplication of SPTFF. The SPTFF step would follow the diafiltration stepin WFT (FIG. 1 ). The SPTFF can concentrate the A1PI to higherconcentrations in WFI than can be achieved with conventional TFF (above100 mg/mL), with only a single pass through the UF membrane assembly atlower pumps speeds, reducing exposure to heat and stress associated withTFF continued pump circulations. The increased flow path length combinedwith a reduced flow rate at higher transmembrane pressure enables higherconcentrations of A1P1 to be achieved [>25% (w/v)] by SPTFF in thepresence of WFI alone. Finally, the concentrated A1PI solution isprecisely diluted to a target concentration [at least 10% (w/v)] with aconcentrated excipient solution at pH adjusted to 7.0 to accomplishosmolality adjustment with either amino acids, sugars, or polyols(represented in Table 3 by alanine, trehalose, and sorbitol,respectively). This process enables high A1PI concentrations to beachieved, while stable formulations of the liquid drug product resultwithout the use of buffers, salts, or surfactants.

TABLE 3 Total protein concentration, specific activity, SE-HPLC,osmolality, and viscosity data for the SPTFF experiments with variousexcipients. Specific Activity Bulk Total (potency/ Sample Protein totalSE-HPLC Osmolality Viscosity Description (mg/mL) protein) AggregateOligomer Monomer (mOsm/kg) (cP) UFDF in 109 1.1 <0.1 3.70 96.27 51 2.9WFI SFTFF in 261 1.1 <0.1 3.96 96.05 249 52.7 WFI 0.12M 202.1 1.0 0.053.96 95.29 292 11.2 Alanine, pH 7.0 0.12M 191 1.2 0.04 3.91 94.83 32313.4 Trebalose, pH 7.0 0.12M 194.2 1.1 0.06 4.03 95.22 307 12.7Sorbitol, pH 7.0

Example 7

pH Range Measured by Zeta Potential

Zeta Potential measurements for A1PI solutions with 0.12 M excipientconcentrations were evaluated across various pHs using a Zetasizer.Higher magnitude of zeta potential (≥40 mV or ≤−40 mV) represents bettercolloidal stability, as molecules that are sufficiently charged tend tobe electrostatically repulsive and are less likely to form aggregates insolution. All A1PI formulations tested (FIG. 5 ) showed colloidalstability within the pH range of 6.6 to 7.4, based on measured ZP valuesbelow −40 mV. In each formulation the zeta potential trended towards 0as the pH approached the A1PI isoelectric point (between 4.0 and 5.0).

Definitions

As used herein, the section headings are for organizational purposesonly and are not to be construed as limiting the described subjectmatter in any way. All literature and similar materials cited in thisapplication, including but not limited to, patents, patent applications,articles, books, treatises, and internet web pages are expresslyincorporated by reference in their entirety for any purpose. Whendefinitions of terms in incorporated references appear to differ fromthe definitions provided in the present teachings, the definitionprovided in the present teachings shall control. It will be appreciatedthat there is an implied “about” prior to the temperatures,concentrations, times, etc. discussed in the present teachings, suchthat slight and insubstantial deviations are within the scope of thepresent teachings herein.

In this application, the use of the singular includes the plural unlessspecifically stated otherwise. Also, the use of “comprise”, “comprises”,“comprising”, “contain”, “contains”, “containing”, “include”,“includes”, and “including” are not intended to be limiting.

As used in this specification and claims, the singular forms “a,” “an”and “the” include plural references unless the content clearly dictatesotherwise.

As used herein, “about” means a quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length thatvaries by as much as 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to areference quantity, level, value, number, frequency, percentage,dimension, size, amount, weight, or length.

Although this disclosure is in the context of certain embodiments andexamples, those skilled in the art will understand that the presentdisclosure extends beyond the specifically disclosed embodiments toother alternative embodiments and/or uses of the embodiments and obviousmodifications and equivalents thereof. In addition, while severalvariations of the embodiments have been shown and described in detail,other modifications, which are within the scope of this disclosure, willbe readily apparent to those of skill in the art based upon thisdisclosure.

It is also contemplated that various combinations or sub-combinations ofthe specific features and aspects of the embodiments may be made andstill fall within the scope of the disclosure. It should be understoodthat various features and aspects of the disclosed embodiments can becombined with, or substituted for, one another in order to form varyingmodes or embodiments of the disclosure. Thus, it is intended that thescope of the present disclosure herein disclosed should not be limitedby the particular disclosed embodiments described above.

It should be understood, however, that this detailed description, whileindicating preferred embodiments of the disclosure, is given by way ofillustration only, since various changes and modifications within thespirit and scope of the disclosure will become apparent, to thoseskilled in the art.

The terminology used in the description presented herein is not intendedto be interpreted in any limited or restrictive manner. Rather, theterminology is simply being utilized in conjunction with a detaileddescription of embodiments of the systems, methods and relatedcomponents. Furthermore, embodiments may comprise several novelfeatures, no single one of which is solely responsible for its desirableattributes or is believed to be essential to practicing the embodimentsherein described.

What is claimed is:
 1. A composition comprising Alpha1-ProteinaseInhibitor (A1PI) in an aqueous solution, wherein the concentration ofA1PI is greater than 200 mg/ml, wherein the composition furthercomprises one or more uncharged excipient.
 2. The composition of claim1, wherein further comprises one or more uncharged excipient.
 3. Thecomposition of claim 2, wherein said one or more uncharged excipient isat a concentration to achieve an osmolality between 220 and 410 mOsm/kgH₂O.
 4. The composition of claim 3, wherein said one or more unchargedexcipient is at a concentration to achieve an osmolality of about 300mOsm/kg H₂O.
 5. A pharmaceutical composition comprising the compositionaccording to claim 1 and a pharmaceutically acceptable carrier.
 6. Thepharmaceutical composition of claim 5, wherein the pharmaceuticalcomposition is formulated for intravenous, subcutaneous, aerosol, orintradermal administration.
 7. The pharmaceutical composition of claim6, wherein the pharmaceutical composition is formulated for subcutaneousadministration.
 8. The pharmaceutical composition of claim 5, whereinthe pharmaceutical composition is encapsulated in nanoparticles.
 9. Thepharmaceutical composition of claim 5, wherein the pharmaceuticalcomposition comprises a time-release polymer.
 10. A method for preparinga composition according to claim 1, comprising a step of preparing asolution of A1PI by concentrating an initial solution of A1PI bysingle-pass tangential flow filtration (SPTFF).
 11. A method forpreparing a composition according to claim 10, wherein said step ofSPTFF is carried out against water for injection (WFI).
 12. A method forpreparing a composition according to claim 10, wherein after the SPTFFstep the solution of A1PI is formulated with one or more unchargedexcipients selected from the group consisting of sorbitol, serine,trehalose, alanine, sucrose, and mannitol, and combinations thereof. 13.A method for preparing a composition according to claim 12, whereinafter the SPTFF step the solution of A1PI is formulated with sorbitol,trehalose, alanine, or a combination thereof.